Kinesins are mechanochemical enzymes which utilize ATP hydrolysis to transport cargos directionally along the microtubule lattice or to regulate microtubule assembly/disassembly. Kinesin function and localization within the cell are tightly regulated via a mechanism of kinase-mediated phosphorylation at specific residues. While the phosphoregulation of kinesins has been studied for decades, analyses have focused almost entirely on phosphorylation sites outside of the conserved catalytic core (motor domain). However, it has recently been shown that the activity of kinesins can be regulated through the phosphorylation of highly conserved residues within their motor domains. This proposal investigates the central hypothesis that phosphorylation of several highly conserved residues within the kinesin motor domain regulates its structural, dynamic and functional interactions with microtubules. Our objectives are to 1) identify new physiologically relevant phosphorylation sites on the kinesin motor domain; 2) identify structural changes induced by phosphorylation at these sites; and 3) determine how these changes are translated into alterations of kinesin catalytic activity and cellular function. The following two Specific Aims will be pursued. Aim 1: Test the hypothesis that regulation of the motor domains of kinesins across the superfamily is mediated through a small number of globally conserved phosphorylation sites. Phosphorylation of these sites rapidly and reversibly alters key structural features of the enzymes' catalytic core. Structure/function relationships that will be tested include tubulin binding and ATP hydrolysis, which are common to all motors. Aim 2: Test the hypothesis that regulation of the motor domains specific to each kinesin family is mediated through a small number of family-conserved phosphorylation sites. Phosphorylation of these sites regulates family-specific functions via specific structural modifications. A multidisciplinary approach to achieve these aims will be pursued, including Electron Paramagnetic Resonance (EPR), single molecule fluorescence spectroscopies, Cryo electron microscopy, biomolecular simulations, multidimensional live cell imaging, and in vitro and in vivo functional analysis. Using a synergistic integration of these techniques, the role of kinesin motor domain phosphorylation will be comprehensively investigated from the molecular to the cellular level. Relevance: The kinesins to be studied in this proposal perform key roles in cell division, development and function, all of which are subject to regulation by phosphorylation. Miss-regulation of these processes has been linked to numerous human diseases. This project is relevant to public health because it will bridge a fundamental gap in our knowledge of kinesin functionality and provide a structural framework to guide the development of novel therapeutic agents targeting these diseases.